1. Field of the Invention
This application relates generally to fluorescence polarization measurements and, in particular, to a method of correcting fluorescence polarization measurements for the effects of background noise.
2. Description of the Related Art
Fluorescence polarization (FP) measurements are used to detect molecular interaction, since molecular interaction typically leads to an increase in the polarization signal. See John C. Owicki, xe2x80x9cFluorescence Polarization and Anisotrophy in High Throughput Screening: Perspectives and Primerxe2x80x9d, JOURNAL OF BIOMOLECULAR SCREENING, Vol. 5, No. 5, 2000 (Hereinafter referred to as xe2x80x9cFP/FA Primerxe2x80x9d, and incorporated by reference) for various examples of assays using fluorescence polarization.
FIG. 1 shows a conventional FP Plate Reader which is used for taking FP measurements from samples in a microtitre plate. Plate 101 contains Samples 105 in Wells 110. Samples 105 are typically comprised of a cell preparation, buffers, a drug or other target substance being tested, and fluorophores that will fluoresce when excited by light. This excitation light is emitted by Lamp 120, filtered by Filter 125, and polarized by Excitation Polarizer 130, before reflecting off of Dichroic Beamsplitter 140 through Objective 145 onto Samples 105. The light emitted by the fluorophores in Samples 105 will proceed through Objective 145 and Dichroic Beamsplitter 140, before being filtered by Filter 150 and analyzed by Emission Polarizer 160. The analyzed emitted light is then detected by Detector 170.
The conventional equation for calculating FP takes the difference between the measured signals when the polarizers are parallel (i.e., when Excitation Polarizer 130 lets light through with the same polarization as light let through Emission Polarizer 160) and when the polarizers are perpendicular (i.e., when Excitation Polarizer 130 lets light through with a polarization orthogonal to the light let through Emission Polarizer 160), and divides this C difference by the sum of the two measurements, as shown by:                               F          ⁢                      xe2x80x83                    ⁢          P                =                  1000          *                                                    I                ∥                            -                              I                ⊥                                                                    I                ∥                            +                              I                ⊥                                                                        (                  Equation          ⁢                      xe2x80x83                    ⁢          1                )            
where I∥ is the intensity measurement with the polarizers parallel, and Ixe2x8axa5 is the intensity measurement with the polarizers perpendicular. The FP signal is a unitless number that indicates degree of fluorescence polarization (DOP), and is typically called millipolarization, xe2x80x9cmilliPxe2x80x9d or xe2x80x9cmPxe2x80x9d.
It is the difference (I∥-Ixe2x8axa5) that carries the assay information, the remaining terms in the equation are for normalization. The object of detection of FP measurements, successful molecular binding events, are indicated by typically small variations in the difference (I∥-Ixe2x8axa5). Because of the need for detecting these small variations, the utility of instruments designed for FP measurement is primarily dependent on the precision of the instrument, and secondarily on the accuracy. In other words, it is more important that the intensity measurements I∥ and Ixe2x8axa5 are precise relative to each other rather than they be individually absolutely accurate.
When making conventional FP measurements using conventional FP measurement devices, one must often choose between accuracy and precision. As an example, consider the problem of background fluorescence in a conventional FP Plate Reader. Background fluorescence is caused by non-probe or non-subject (the subject being the molecule under study) elements, such as Wells 110 or the cell preparation and buffers in Samples 105, which fluoresce and add to the emitted light which reaches Detector 170. This can affect the accuracy of the FP measurements. The accuracy problem can be illustrated by the following hypothetical situation, where a xe2x80x9cpurexe2x80x9d FP measurement (meaning xe2x80x9cpurelyxe2x80x9d from the fluorophores or probes) is sought:                               F          ⁢                      xe2x80x83                    ⁢                      P            probe                          =                  1000          *                                                    I                ∥                probe                            -                              I                ⊥                probe                                                                    I                ∥                probe                            +                              I                ⊥                probe                                                                        (                  Equation          ⁢                      xe2x80x83                    ⁢          2                )            
where
I∥probe=the intensity signal of parallel polarized light from the probes; and
Ixe2x8axa5probe=the intensity signal of perpendicularly polarized light from the probes.
In our hypothetical, we will assume that I∥probe=60,000 and Ixe2x8axa5probe=40,000, so the actual FP of the probes, FPprobe,=200 mP. Furthermore, we will assume that the intensity signal of the background is equal to some proportion of the total intensity signal of the probes:
Ibkgrnd=Ibkgrnd::probe*Iprobe 
where
Ibkgrnd=total intensity signal of the background;
Ibkgrnd::probe=the ratio of background signal to probe signal; and
Iprobe=total intensity signal of the probes.
A reasonable assumed value for Ibkgrnd::probe is ⅓ or 0.33, which will result in Ibkgrnd=33,000. Now, we will determine the parallel and perpendicularly polarized components of the background signal:       I    ∥    bkgrnd    =            I      bkgrnd        *                  (                  1          +                      FP            bkgrnd                          )            2000      xe2x80x83Ixe2x8axa5bkgrnd=Ibkgrndxe2x88x92I∥bkgrnd 
where
I∥bkgrnd=the intensity of the parallel polarized component of the background;
FPbkgrnd=the fluorescence polarization of the background; and
Ixe2x8axa5bkgrnd=the intensity of the perpendicularly polarized component of the background.
Assuming a value of 450 mP for the background FP (FPbkgrnd), the parallel polarized component of the background, I∥bkgrnd, equals 23,930 and the perpendicularly polarized component, Ixe2x8axa5bkgrnd, equals 9075. The total intensity of the polarized signal received by Detector 170 equals the combination of the probe signal and the background signal:
Ixe2x8axa5meas=Ixe2x8axa5bkgrnd+Ixe2x8axa5probe 
Ixe2x8axa5meas=83,930 
xe2x80x83I∥meas=I∥bkgrnd+I∥probe 
I∥meas=9075 
In the end, the final measured FP as calculated by the intensity measurements at the detector is:                               F          ⁢                      xe2x80x83                    ⁢                      P            meas                          =                  1000          *                                                    I                ∥                meas                            -                              I                ⊥                meas                                                                    I                ∥                meas                            +                              I                ⊥                meas                                                                        (                  Equation          ⁢                      xe2x80x83                    ⁢          3                )            xe2x80x83FPmeas=262
Thus, the measured FP of 262 is more than 25% in error (from the real FP of 200). If this type of error margin is unacceptable to the experimenter, she may use a method to compensate for the signal noise generated by the background. Such methods have been developed over time, as discussed in FP/FA Primer. These conventional methods to compensate for background fluorescence use a subtractive approach to decrease the background signal source. For example, Equation 4 below directly subtracts parallel and perpendicular measurements of background wells (containing one or more assay components, but not the fluorophore) from the parallel and perpendicular measurements of the sample wells under study (all assay materials including fluorophore):                               F          ⁢                      xe2x80x83                    ⁢                      P            meas                          =                  1000          *                                                    (                                                      I                    ∥                    meas                                    -                                      I                    ∥                    bkgrnd                                                  )                            -                              (                                                      I                    ⊥                    meas                                    -                                      I                    ⊥                    bkgrnd                                                  )                                                                    (                                                      I                    ∥                    meas                                    -                                      I                    ∥                    bkgrnd                                                  )                            +                              (                                                      I                    ⊥                    meas                                    -                                      I                    ⊥                    bkgrnd                                                  )                                                                        (                  Equation          ⁢                      xe2x80x83                    ⁢          4                )            
The disadvantage of this approach is that the accuracy of background correction is subject to changes in excitation lamp intensity, well-to-well variations in sampled volume and meniscus, and the degree of photobleaching between the background measurement and the sample measurement. In effect, the background correction step injects into the calculation additional noise, which reduces measurement precision. Thus, in order to achieve greater accuracy in one""s FP measurements, one may end up sacrificing the precision of such measurements. But, as is discussed above, this is not desirable, since the precision of these measurements relative to each other is more important than the accuracy of the individual measurements.
Some experimenters may prefer losing the accuracy of their measurements, i.e., by not performing any background compensation, in order to save the precision of the measurements. However, this solution (of not performing any background correction) is also not desirable because of the effects of such a loss of accuracy when making comparisons between different experiments, assays, samples, etc. For example, part of the job of assay development technicians is to compare assays from different experiments; but this is made more difficult (and the results are more questionable) when the accuracy of the measurements from the different experiments is problematic.
A particularly problematic situation is when the background signal is a substantial portion of the total signal, say 25% or more. This typically occurs when tracer concentrations are reduced to less than a few hundred picoMolar (pM). For example, if
a) the effective sampled volumes vary from well to well by 5%,
b) 25% of the measured fluorescence emission signal is background fluorescence,
c) the background FP signal is 450 mP, and
d) the actual sample FP signal is 150 mP,
the degradation of the instrument measurement, as represented by standard deviations in multiple FP measurements, due to background compensation alone would be 5 mP. This limitation may be the most significant barrier to performing assays with tracer concentrations less than 100 pM. When dealing with such small amounts, not using any background compensation is also unsatisfactory, because the effect of the background noise becomes so large in comparison to the real (probe) signal that both the precision and accuracy of the FP measurements are substantially degraded. This is a crucial problem because many assays should be operated at such low levels, near the Kd point.
Thus, there is a need for a background correction scheme that does not add noise into the FP measurement calculation, and that enables background-corrected measurement with the measurement of the actual sample providing the only source of noise. Furthermore, there is a need for a more precise method of correcting FP measurements for the effects of background fluorescence, where the background fluorescence correction does not inject the noise or uncertainty of the conventional method into the FP measurement. This need is particularly acute when fluorophore concentrations get low (e.g., below a few hundred pM), which causes significant error in both the precision and accuracy of FP measurements in the conventional method.
One object of the present invention is to provide a background correction scheme that does not add noise into the FP measurement calculation, and that enables background-corrected measurement with the measurement of the actual sample providing the only source of noise.
Another object of the present invention is to provide a more precise method of correcting FP measurements for the effects of background fluorescence, where the background fluorescence correction does not inject the noise or uncertainty of the conventional method into the FP measurement.
Yet another object of the present invention is to provide a correction method which does not introduce a significant source of error when fluorophore concentrations get low (e.g., below a few hundred pM).
Yet another object of the present invention is to provide a method which confers greater precision and accuracy on FP measurements than conventional methods when fluorophore concentrations get low (e.g., below a few hundred pM).
Still another object of the present invention is to provide more accurate fluorescence polarization measurements without necessarily degrading the instrument signal-to-noise ratio.
These and other objects are achieved by the present invention in which a multiplicative ratio approach is used to remove the effects of the unwanted background fluorescence rather than the conventional subtractive approach, thus preserving both the precision and accuracy of the FP measurements. The method according to the present invention, comprises selecting an appropriate multiplicative ratio is selected, then the selected multiplicative ratio is calculated using measurements from samples. The calculated multiplicative ratio is multiplied by an appropriate value in one of the standard FP measurement equations (Equations 1 or 4) or an appropriate value in an equation derived from one of the standard FP measurement equations. After (or during) this calculation, the corrected FP measurement is calculated. When such multiplicative ratios are applied to the appropriate value or values in an FP measurement equation, the effects of background noise can be reduced without decreasing the precision of the FP measurements.
In a first presently preferred embodiment of the present invention, a multiplicative ratio comprised of the relative proportion of light from a background well fluorescence emission to light from a sample well fluorescence emission is calculated, and is appropriately multiplied into the fluorescence polarization calculation, resulting in a corrected FP measurement that is both accurate and precise. In a second presently preferred embodiment, a multiplicative ratio comprised of the total intensity of the first sample divided by the total intensity of the current sample is calculated, and is appropriately multiplied by the first measured background intensity, resulting in a corrected background intensity, which is used in a corrected FP measurement equation that is both accurate and precise.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.